Macrophages are one of the effector cell types that play an important role in immunosurveillance against neoplastic growth in vivo. In vitro, cell-mediated cytotoxicity requires selective binding between activated macrophages and target cells as well as the concomitant release of cytotoxic factors. Some of the cytotoxic factors secreted by activated macrophages include reactive oxygen species such as the superoxide anion and hydrogen peroxide, arginase, interleukin 1, and tumor necrosis factor-.alpha. (TNF-.alpha.). Acquired resistance to the toxic effects of these factors by tumor cells could be one mechanism which leads to the onset and spread of tumor formation in vivo.
The observation that TNF-.alpha. can act as a potent effector of the macrophage-mediated antitumor response provides a rationale for its use in further studies on the regulation of tumorigenesis in vivo and tumor cell growth in vitro. The genes encoding TNF-.alpha. and TNF-.beta., a structurally related cytotoxic protein formerly known as lymphotoxin, have been cloned and the corresponding proteins expressed in Escherichia coli. These proteins display an array of biological activities, including induction of hemorrhagic necrosis of Meth A sarcomas in vivo, inhibition of the growth of certain tumor cells in vitro, synergistic enhancement of the in vitro anticellular effects of IFN-.gamma., activation of human polymorphonuclear neutrophil functions, and inhibition of lipid biosynthesis. Recently, rHuTNF-.alpha. was shown to augment the growth of normal diploid fibroblasts in vitro. The divergent proliferative responses in the presence of rHuTNF-.alpha. are sometimes related to variations in TNF binding.
Growth factors and their receptors are involved in the regulation of cell proliferation and they also appear to play a key role in oncogenesis. Of the known proto-oncogenes, three are related to a growth factor or a growth factor receptor. These genes include c-sis, which is homologous to the transforming gene of the simian sarcoma virus and is the B chain of platelet-derived growth factor (PDGF); c-fms, which is homologous to the transforming gene of the feline sarcoma virus and is closely related to the macrophage colony-stimulating factor receptor (CSF-1R); and c-erbB, which encodes the EGF receptor (EGFR) and is homologous to the transforming gene of the avian erythroblastosis virus (v-erbB). The two receptor-related proto-oncogenes, c-fms and c-erbB, are members of the tyrosine-specific protein kinase family to which many proto-oncogenes belong.
Recently, a novel transforming gene was identified as a result of transfection studies with DNA from chemically induced rat neuroblastomas. This gene, called neu, was shown to be related to, but distinct from, the c-erbB proto-oncogene. By means of v-erbB and human EGFR as probes to screen human genomic and complementary DNA (cDNA) libraries, two other groups independently isolated human erbB-related genes that they called HER2 and c-erbB-2 respectively. Subsequent sequence analysis and chromosomal mapping studies revealed that c-erbB-2, and HER2 are species variants of neu. A fourth group, also using v-erbB as a probe, identified the same gene in a mammary carcinoma cell line, MAC 117, where it was found to be amplified five- to ten-fold.
This gene, which will be referred to herein as HER2, encodes a new member of the tyrosine kinase family; and is closely related to, but distinct from, the EGFR gene as reported by Coussens et al., Science 230, 1132 (1985). HER2 differs from EGFR in that it is found on band q21 of chromosome 17, as compared to band p11-p13 of chromosome 7, where the EGFR gene is located. Also, the HER2 gene generates a messenger RNA (mRNA) of 4.8 kb, which differs from the 5.8- and 10-kb transcripts for the EGFR gene. Finally, the protein encoded by the HER2 gene is 185,000 daltons, as compared to the 170,000-dalton protein encoded by the EGFR gene. Conversely, on the basis of sequence data, HER2 is more closely related to the EGFR gene than to other members of the tyrosine kinase family. Like the EGFR protein, the HER2 protein (p185) has an extracellular domain, a transmembrane domain that includes two cysteine-rich repeat clusters, and an intracellular kinase domain, indicating that it is likely to be a cellular receptor for an as yet unidentified ligand. HER2 p185 is referred to as p185 or the HER2 receptor herein.
Southern analysis of primary human tumors and established tumor-derived cell lines revealed amplification and in some cases rearrangement of the EGF receptor gene. Amplification was particularly apparent in squamous carcinomas and glioblastomas. The HER2 gene was also found to be amplified in a human salivary gland adenocarcinoma, a renal adenocarcinoma, a mammary gland carcinoma, and a gastric cancer cell line. Recently, Slamon et al., Science 235, 177 (1987) demonstrated that about 30% of primary human breast carcinoma tumors contained an amplified HER2 gene. Although a few sequence rearrangements were detected, in most tumors there were no obvious differences between amplified and normal HER2 genes. Furthermore, amplification of the HER2 gene correlated significantly with the negative prognosis of the disease and the probability of relapse.
To investigate the significance of the correlation between overexpression and cellular transformation as it has been observed for proto-oncogenes c-mos and N-myc, a HER2 expression vector and a selection scheme that permitted sequence amplification after transfection of mouse NIH 3T3 cells was employed by Hudziak et al., Proc. Natl. Acad, Sci. (USA) 84, 7159 (1987). Amplification of the unaltered HER2 gene in NIH 3T3 cells lead to overexpression of p185 as well as cellular transformation and tumor formation in athymic mice.
The effects of antibodies specifically binding growth factors or growth factor receptors has been studied. Examples are discussed below.
Rosenthal et al., Cell 46, 301 (1986) introduced a human TGF-.alpha. cDNA expression vector into established non-transformed rat fibroblast cells. Synthesis and secretion of TGF-.alpha. by these cells resulted in loss of anchorage-dependent growth and induced tumor formation in nude mice. Anti-human TGF-.alpha. monoclonal antibodies prevented the rat cells from forming colonies in soft agar, i.e. loss of anchorage dependence. Gill et al. in J. Biol. Chem. 259, 7755 (1984) disclose monoclonal antibodies specific for EGF receptor which were inhibitors of EGF binding and antagonists of EGF-stimulated tyrosine protein kinase activity.
Drebin et al. in Cell 41, 695 (1985) demonstrated that exposure of a neu-oncogene-transformed NIH 3T3 cell to monoclonal antibodies reactive with the neu gene product, cause the neu-transformed NIH 3T3 cell to revert to a non-transformed phenotype as determined by anchorage independent growth. Drebin et al. in Proc. Natl. Acad. Sci. 83, 9129 (1986) demonstrated that in vivo treatment with a monoclonal antibody (IgG2a isotype) specifically binding the protein encoded by the neu oncogene significantly inhibited the tumorigenic growth of neu-transformed NIH 3T3 cells implanted into nude mice.
Akiyama et al. in Science 232, 1644 (1986) raised antibodies against a synthetic peptide corresponding to 14 amino acid residues at the carboxy-terminus of the protein deduced from the c-erbB-2 (HER2) nucleotide sequence.
Growth factors have been reported to interact in both a synergistic and an antagonistic manner. For example, TGF-.alpha. and TGF-.beta. synergistically enhance the growth of NRK-49F fibroblasts, whereas PDGF down regulates EGF receptor function on 3T3 cells. A variety of transformed cells secrete factors which are believed to stimulate growth by an autocrine mechanism. Sugarman et al., Cancer Res. 47, 780 (1987) demonstrated that under certain conditions, growth factors can block the antiproliferative effects of TNF-.alpha. on sensitive tumor cells. Specifically, epidermal growth factor (EGF) and recombinant human transforming growth factor-.alpha. (rHuTGF-.alpha.) were shown to interfere with the in vitro antiproliferative effects of recombinant human tumor necrosis factor-.alpha. (rHuTNF-.alpha.) and -.beta. on a human cervical carcinoma cell line, ME-180. The inhibitory effect could be observed at EGF or rHuTGF-.alpha. concentrations of 0.1 to 100 ng/ml, and was maximal between 1 and 10 ng/ml. This response was apparently not due to down regulation of the TNF receptor or to alteration of the affinity of TNF-.alpha. for its receptor. Since the antiproliferative effect of recombinant human interferon-.gamma. was not significantly affected by the presence of EGF or rHuTGF-.alpha., the inhibition was specific for recombinant TNFs and was not due solely to enhanced proliferation induced by the growth factors. Neither growth factor had a substantial protective effect on the synergistic cytotoxicity observed when tumor cells were exposed simultaneously to rHuTNF-.alpha. and recombinant human interferon-.gamma.. TGF-.beta. can also interfere with the antiproliferative effects of rHuTNF-.alpha. in vitro. At concentrations of less than 1 ng/ml, TGF-.beta. significantly antagonized the cytotoxic effects of rHuTNF-.alpha. on NIH 3T3 fibroblasts. Since EGF, platelet-derived growth factor, and TGF-.beta. all enhanced NIH 3T3 cell proliferation, but only TGF-.beta. interfered with rHuTNF-.alpha. cytotoxicity, the protective effects of TGF-.beta. were not related in a simple manner to enhanced cell proliferation. rHuTGF-.alpha. and TGF-.beta. did not have a significant protective effect against rHuTNF-.alpha.-mediated cytotoxicity on two other tumor cell lines, BT-20 and L-929 cells.
It is an object of the subject invention to provide antibodies capable of inhibiting growth factor receptor function.
It is a further object of the invention to provide an improved assay for the HER2 receptor.
It is a further object of the invention to provide improved methods of tumor therapy.
It is a further object of the invention to provide a method of inhibiting the growth of tumor cells which overexpress a growth factor receptor and/or growth factor.
It is a further object of the invention to provide a method for treating a tumor by treatment of the tumor cells with antibodies capable of inhibiting growth factor receptor function, and with cytotoxic factors such as tumor necrosis factor.
A still further object of the invention is to provide an assay for tyrosine kinases that may have a role in tumorigenesis.
Other objects, features and characterisitics of the present invention will become apparent upon consideration of the following description and the appended claims.